Necrotizing enterocolitis (NEC), a disease characterized by rapid development of intestinal inflammation, often resulting in gut necrosis, is a leading cause of neonatal morbidity and mortality. NEC pathogenesis is poorly understood, and there are no diagnostic tests to predict which infants will develop NEC; furthermore, there are no effective prevention strategies. Recent studies have identified red blood cell (RBC) transfusion, a common therapy in extremely low birth weight (ELBW) infants, as an important risk factor and potential causative factor in the development of transfusion-related NEC (TR-NEC), defined as NEC developing within 48 hours of a RBC transfusion. However, biologic and clinical determinants of RBC TR-NEC remain poorly understood. Our proposed study will enroll 220 ELBW infants and be the first prospective, large, multicenter cohort study to investigate the pathophysiologic mechanisms that underlie the development of NEC following RBC transfusion. Our overarching hypothesis is that irradiation of RBC units followed by prolonged storage perturbs RBC metabolism/function, leading to paradoxical microvascular vasoconstriction, tissue hypoxia and TR-NEC in transfused infants with already impaired gut oxygenation due to significant anemia. Our three specific aims will test key aspects of this hypothesis. This study will utilize innovative methods to study TR-NEC, including metabolomics (Aims 1 and 2), near-infrared spectroscopy (NIRS) which is an in vivo non-invasive measure of mesenteric regional saturation of oxygen (MES-rSO2; Aims 1 and 3), and in vitro RBC functional studies (Aim 2). This proposed study will fill key knowledge gaps regarding the effects of RBC storage time and irradiation on neonatal gut injury. Further, these data will guide development of new interventions, such as using NIRS to identify susceptible infants, limiting IST of stored RBC products, and using metabolite biomarkers to predict RBC safety. This project addresses the P01 central theme since ELBW preterm infants are a population vulnerable to the development of TR-NEC with antecedent RBC transfusion(s). This project interacts with Projects 1, 3, and 4 by identifying metabolic biomarkers that are associated with adverse transfusion events. Additionally Core A (regulatory approvals, sample repository), Core B (metabolomics), and Core C (biostatistics) are utilized heavily in the proposed studies.